Tetraguaiacol Reaction

Introduction:
Many vital chemical reactions do not naturally happen at fast enough rates to maintain life. Enzymes are a type of protein that speed up these reactions because they are catalytic, meaning they increase the rate of the reaction without being consumed by it (Freeman, 2014, p. 54). In 1894, Emil Fischer proposed the “lock-and-key” method in order to explain how enzymes work. As the name suggests, the enzyme represents the lock as the substrate represents the key. A substrate is the reactant the enzyme binds to that the enzyme then quickly transforms into a product (Campbell, 2001, p. 151). The substrate only binds to a particular region of the enzyme called the active site. At the active site, hydrogen bonds or other weaker intermolecular

forces interact with the amino acids encompassing the substrate and orientating it correctly. Once bonded, the R-groups of the amino acids in the active site stabilize the transition state of the reaction, which, in turn, lowers the activation energy of the reaction therefore speeding it up (Freeman, 2014, p. 54).
Generally, as the temperature increases, so does the rate of the reaction. This is because more temperature means more kinetic energy, which means more movement, which means more substrate molecules colliding and entering the active sites of enzymes, which means more products being formed. Eventually, the reaction will reach its optimal temperature (where the enzyme is functioning most quickly and efficiently), and if the temperature keeps increasing past this point, the rate of the reaction will decrease tremendously. This is because after the optimal temperature is surpassed, the bonds in the active sites of enzymes destabilize and then the enzyme ultimately denatures. Denaturation alters the shape of the original protein as it loses its structure due to stressful conditions, which in this case means extreme heat (Campbell, 2001, p. 154).
Inhibitors also have an effect on the activity of enzymes. In a type of enzyme inhibition called competitive inhibition, inhibitor molecules mimic the shape of the actual substrate molecules and block their entrance into the active sites of enzymes, thus, deceasing the rate of the reaction since the substrate molecules cannot even enter the active sites (Freeman, 2014, p. 54).
In both experiments, the type of enzyme being used is called peroxidase whose main job is to rid the body of the substrate hydrogen peroxide, which can cause severe cell damage.

Hydrogen peroxide is a toxic by-product of many crucial metabolic reactions for aerobic organisms. Lastly, a colorless dye called guaiacol is added to the reaction so that it can bind to peroxidase, get oxidized while hydrogen peroxide is reduced to water, and form a brown tetraguaiacol. That is an example of an oxidation-reduction reaction, which can be monitored using a spectrophotometer due to the brown nature of tetraguaiacol. With a LabQuest attached to a spectrophotometer, absorbance of the enzyme/substrate solution is monitored versus time when exposed to different conditions.
In the first experiment, temperature affects the rate enzyme activity, and the more temperature increases, the faster the reaction will proceed until the optimum temperature is reached. After that, enzyme activity will decrease. Furthermore, hydroxylamine and hydrogen peroxide have extremely similar structures. In the second experiment, hydroxylamine affects peroxidase’s activity. The more hydroxylamine, the less enzyme activity will occur due to the competitive inhibition of the

hydroxylamine.
Methods:
In part A of the experiment, the spectrophotometer was calibrated to take absorbance readings for 500 nm every 20 seconds for a length of 120 seconds per sample.

In part B of the experiment, the amount of peroxidase to use for the rest of the experiment was standardized, and it was concluded that 250μl of peroxidase should be used in the experiment for optimal results. In part C of the experiment, the effect of temperature was tested. Six cuvettes were numbered, and three mL of peroxidase was added to a test tube to then be placed in a hot/boiling water bath for 15 minutes. After the test tube was taken out and let cooled, 250μl of the boiled water was added to cuvette six. Cuvette two was placed in ice, cuvette four was placed in the 32°C water bath, and cuvette five was placed in a 48°C water bath for ten minutes. After the ten minutes was up, 1000μl of buffer was added to cuvettes one through six, 500 mL of guaiacol was added to all cuvettes as well, and 250mL of normal peroxidase was added to cuvettes two through five. Cuvette one was used to calibrate the spectrophotometer. All of these values can be seen in the table

below.
Cuvette one was ran first to calibrate/blank the spectrophotometer. Then, 500μl of hydrogen peroxide was added to cuvette two while simultaneously starting to collect absorbance data on the spectrophotometer for 120 seconds. That was done for cuvettes three through six as well. All absorbencies and the linear slope of the absorbencies were recorded in Table 3. Lastly, the solutions were disposed in a waste container under a fume hood, and the cuvettes were rinsed with ethanol.
In part D of the experiment, the effect of inhibition on enzyme activity was tested. First, 500μl of 2% hydroxylamine was added to 1000μl of peroxidase, mixed and let set for 15 minutes. This was the “Hydroxylamine Treated Extract.” Three test tubes were then numbered and filled with the proper amount of reagents as found in table 4.
Cuvette one was ran first to calibrate/blank the spectrophotometer. Then, 500μl of hydrogen peroxide was added to cuvette two while simultaneously starting to collect absorbance data on the spectrophotometer for 120 seconds. That was done for cuvettes three as well. All absorbencies and the linear slope of the absorbencies were recorded in Table 5. Finally, the solutions were disposed in a waste container under a fume hood, and the cuvettes were rinsed with ethanol just like before. Lastly though, the percent inhibition was calculated by the following formula: % inhibition = [(normal enzyme activity - inhibited enzyme activity) / (normal enzyme activity)] * 100%
Results:
In this lab, there are two experiments addressing what affects enzymatic activities. In the first experiment, the effect of temperature on enzymatic activity was tested. As seen in Figure 3, the results showed that as the temperature increased, the enzymatic activities decreased, which is theoretically not true and will be discussed in the next section.

In the second experiment, the effect of hydroxylamine, a competitive inhibitor, on the peroxidase enzymatic activities was tested. As seen in Figure 5, the results showed that the hydroxylamine successfully inhibited peroxidase’s enzymatic activities. Additionally, the percent of inhibition was 98.862%, which is very high.
% inhibition = [(2.4435 x 10-4 - 2.7803 x 10-6) / (2.4435 x 10-4)] * 100% = 98.862%
Discussion:
In experiment one, the data did not support the hypothesis or any of the literature. The hypothesis was that as the temperature increased, so would the enzymatic activity up to the optimum temperature and then it would drop significantly. As indicated by Figure 3, the enzymatic activity decreased as the temperature increased. So unless peroxidase’s optimum temperature is zero, which is highly unlikely, human error has caused some discrepancy in the results. Perhaps the spectrophotometer was not calibrated correctly or the solutions were not mixed properly. Additionally, cross-contamination between solutions could have occurred as well. Because the higher the temperature, the more kinetic movement molecules have the more chances they have to enter an active site, so it wouldn’t make any sense unless human error was involved (Campbell, 2001, p. 154).
In experiment two, on the other hand, the results supported the hypothesis. The hydroxylase and the hydroxylamine molecules have very similar structures therefore inhibiting the hydroxylase from always entering the active site because sometimes hydroxylamine is there instead (Dolphin, 1997, p. 6). That was a prime exactly of competitive inhibition where the active site for peroxidase was completely blocked off therefore slowing the rate of enzymatic activity down as seen in Figure 5. Lastly, as seen by the formula in the results, the percent of inhibition was 98.862%, which indicates the success rate of the competitive inhibitor.